Calcium hunger in the parathyroidectomized rat is specific.

Calcium ingestion consequent on a challenge to calcium homeostasis such as parathyroidectomy or calcium deficient diet often appears to be nonspecific: rats rendered calcium deficient by a calcium-deficient diet increase ingestion of many tastants besides calcium, including MgCl2 and in particular, NaCl. Rats rendered calcium deficient by parathyroidectomy (PTX) have also been reported to increase ingestion of magnesium. Thus, magnesium, a chemically related molecule, and NaCl, a preferred mineral, pose challenges to the notion of a specific calcium hunger in calcium-deficient rats. Here, we reexamine the specificity of calcium preference of rats rendered calcium hungry by PTX by allowing them to choose between CaCl2 and MgCl2, and between CaCl2 and NaCl. We find that PTX rats clearly prefer calcium to either tastant. A review of the literature on tastant selection by calcium-hungry rats suggests that, given the choice between calcium and other tastants, they prefer calcium. Our findings lend credence to the notion of a calcium specific appetite in calcium-deficient rats.     

Effect of parathyroid hormone on renal calcium and magnesium reabsorption in magnesium deficient rats

Tubular magnesium reabsorption was investigated by recollection micropuncture and in vivo microperfusion techniques in acutely thyropara-thyroidectomized rats made magnesium deficient by dietary deprivation. Henle's loop which normally reclaims the major portion of filtered magnesium was examined by elevation of intraluminal magnesium concentration. The transport capacity in these conditions was significantly lower in magnesium deficient rats (41%) compared to normal animals (71%) at comparable magnesium delivery rates. Acute infusion of MgCl₂ further depressed loop magnesium reabsorption independent of intraluminal magnesium delivery. Parathyroid hormone did not alter magnesium transport capacity in magnesium deficient rats but resulted in enhanced transport in acutely hypermagnesemic deficient rats. Calcium reabsorption followed a similar qualitative pattern as magnesium with respect to loop function and urinary excretion. These results are consistent with a depressed transport capacity for magnesium in the loop of Henle of magnesium deficient rats which is independent of intraluminal magnesium delivery and circulating parathyroid hormone level.     

High fructose feeding of magnesium deficient rats is associated with increased plasma triglyceride concentration and increased oxidative stress.

The purpose of this study was to assess whether dietary carbohydrate could differentially influence the consequences of magnesium deficiency with particular emphasis on lipid metabolism and oxidative stress. Rats were fed a sucrose based or starch based diet either adequate or deficient in magnesium for two weeks. Magnesium deficient rats, as compared with rats fed magnesium adequate diets, displayed the usual decrease in plasma magnesium concentration. The classic symptoms of inflammation including hyperaemia, increased number of blood leukocytes and enlarged spleen weight were observed in these rats. Plasma TG and plasma apo B concentrations were also significantly increased. In addition, magnesium-deficient animals presented an increased susceptibility to lipid peroxidation of heart and liver tissues as shown by TBARS concentration. Regardless of magnesium status, sucrose feeding did not affect the magnesium plasma level and inflammatory parameters. Feeding rats the sucrose diets induced hypertriglyceridaemia and increased plasma apo B concentration. Heart and liver susceptibility to lipid peroxidation were significantly increased in rats fed the sucrose diets as compared with those fed the starch diets. Sucrose feeding in magnesium deficient rats was associated with higher plasma triglycerides concentration and higher tissue susceptibility to peroxidation as compared with magnesium deficient rats fed the starch diet. The results emphasised the potential detrimental and additional effect of sucrose feeding and magnesium deficiency on cardiovascular risk. Since the intake of magnesium has been reduced appreciably in industrialised countries while fructose consumption has been rapidly increased, the impact of this eating pattern should be clarified in humans.     

Effect of nutritional copper deficiency on adjuvant arthritis and immunocompetence in the rat

Both severe and marginal copper deficiency were produced in male Sprague Dawley rats prior to induction of adjuvant arthritis. Degree of copper deficiency was confirmed by analysis of plasma, liver, and brain samples prior to adjuvant injection. Incidence of adjuvant arthritis was the same in both copper deficient and control animals although the severity was slightly but not statistically less in the former. However, recovery from foot edema was impaired in copper-deficient animals, while marginally copper-deficient animals recovered at the same rate as did controls. Plasma copper concentration increase in response to the injection of adjuvant and the increase was directly related to dietary copper content. Plasma zinc concentration was decreased in arthritic animals and the decrease was inversely correlated to paw edema. Liver copper, zinc, and iron concentrations in arthritic animals remained unchanged or increased slightly in comparison to the corresponding non-injected controls. Copper-deficient rats were immunosuppressed as demonstrated by impaired responsiveness to the T-cell dependent contact sesitizing agent oxazolone and diminshed capacity to respond to the T-cell independent antigen Type III pneumococcal polysaccharide. Although a statistical difference in paw volumes was not found for group of animals fed diets differing in copper content, it is postulated that copper deficiency may alter the severity and kinetics of adjuvant arthritis by impairing aspects of the immune response and the tissue repair processes subsequent to injury.     

Zinc deficiency and behavior: A developmental perspective

Zinc deficiency was induced in 35 and 300 day-old male Holtzman rats. Group ZDA was fed ad lib a diet deficient in zinc (1 ppm), ZSP controls were pair-fed a diet supplemented with zinc (50 ppm) and ZSA controls were fed ad lib a diet supplemented with zinc (50 ppm). Physical status and six open-field behaviors were evaluated. Food intake, body weight and plasma zinc concentrations were significantly reduced in both age groups. Expanded use of the open-field revealed significantly lower latencies to explore the novel environment and significantly lower ambulation scores in the young and older zinc deficient rats. Older rats spent significantly less time grooming than their controls. Rearing was significantly less in young zinc deficient rats and “kangaroo-like” posture was evident. Young rats made deficient during critical periods of growth and development, were at greater risk for most parameters tested, compared to the older deficient rats. These results do demonstrate, however, that feeding low-zinc diets to older, fully developed animals results in significant physical and behavioral impairment.     

Effect of magnesium deficiency on 15-hydroxyeicosatetraenoic acid in cultured human umbilical arterial endothelial cells.

Effects of magnesium deficiency on the production of 15-hydroxyeicosatetraenoic acid (15-HETE) and cytosolic free calcium concentration in human umbilical arterial endothelial cells were studied by radioimmunoassay. 15-HETE release by endothelial cells incubated with normal magnesium media (900 microM Mg2+) for 24 h was 2.2 +/- 0.3 ng/mg protein. 15-HETE release gradually increased in proportion to decrease of magnesium. Low magnesium media (180 microM Mg2+) caused an increase in 15-HETE release in a time-dependent manner. Cytosolic free calcium concentration of endothelial cells in normal magnesium media fluctuated between 129.4 nM and 134.2 nM during a 24 h period. Low magnesium media (180 microM Mg2+) caused a time-dependent rise in cytosolic free calcium concentration which is consistent with a time-dependent increase in H-HETE release. High magnesium medium (1800 microM Mg2+) did not have any effect on cytosolic free calcium concentration or 15-HETE production. In conclusion, 15-HETE release by endothelial cells was stimulated by increase in cytosolic free calcium concentration induced by magnesium deficient media. It is suggested that magnesium deficiency induces atherosclerosis via increase in 15-HETE production.     

Towards bio monitoring of toxic (lead) and essential elements in whole blood from 1- to 72-month old children: a cross-sectional study

Objectives

Minerals such as zinc, copper, selenium, calcium, and magnesium are essential for normal human development and functioning of the body. They have been found to play important roles in immuno-physiologic functions. The study is to evaluate the distribution and correlation of nonessential (lead) and essential elements in whole blood from 1- to 72-month old children.

Methods

The cross-sectional study was performed in 1551 children. Six element concentrations, including copper (Cu), zinc (Zn), calcium (Ca), magnesium (Mg), iron (Fe) and lead (Pb) in the blood were determined by atomic absorption spectrometry. Distributions and correlations of trace elements in different age groups were analyzed and compared. A Pearson correlation controlled for age and gender was used to assess the relationship of non essential (lead) and essential elements.

Results

Levels of copper and magnesium were 18.09 ± 4.42 µmol/L and 1.42 ± 0.12 mmol/L, respectively. 6.04% of all children showed copper levels below the normal threshold, the levels of Magnesium were stable in different age groups. Though the overall mean blood zinc and iron concentrations (61.19 ± 11.30 µmol/L and 8.24 ± 0.59 mmol/L, respectively) gradually increased with age and the overall deficiency levels (24.1% and 36.0%, respectively) decreased with age, zinc and iron deficiencies were still very stable. Controlling for gender and age, significant positive correlations were found when comparing copper to zinc, calcium, magnesium, and iron ((r = 0.333, 0.241, 0.417, 0.314 ,p < 0.01); zinc to magnesium and iron (r = 0.440, 0.497p < 0.01); and magnesium to Calcium and iron (r = 0.349, 0.645, p < 0.01). The overall mean blood lead levels (41.16 ± 16.10) were relatively unstable among different age groups. The prevalence of lead intoxication in all children was 1.3% .Calcium levels decreased gradually with age, with an overall concentration of 1.78 ± 0.13 mmol/L.

Conclusion

Significant negative correlations were also noted between Pb and Zn, Fe (r = −0.179, −0.124.p < 0.01) .The importance of calcium deficiency and supplementation is well realized, but the severity of iron and zinc deficiency is not well recorded. The degree of lead intoxication in all the children studied was low; The established reference intervals for Cu, Zn, Ca and Mg provide an important guidance for the reasonable supplementation of essential elements during different age groups.     

Activity and t-maze performance in iron deficient rats

Iron deficient rats were tested in a t-maze to see whether the changes in activity during the onset and progress of the deficiency would affect their performance in the maze. Increased exploratory activity in the initial stages of iron deficiency was reflected in enhanced t-maze learning, whereas the lower activity seen as the deficiency progressed was associated with normal maze-learning. The t-maze task may not be sufficiently sensitive to pick up differences between deficient and control animals due to reduced general activity in the later stages of iron deficiency.     

Early-onset increased calcium and decreased magnesium concentrations and an increased calcium/magnesium ratio in SHR versus WKY.

Alterations in the metabolism of calcium and magnesium have been implicated in the pathogenesis of primary hypertension. Calcium influx across the external cellular membrane in smooth muscle cells and cardiomyocytes plays a crucial role in the control of cellular excitation contraction and impulse propagation. Intracellular calcium and magnesium concentrations are controlled by reversible binding to specific calcium binding proteins. The calcium and magnesium flux across the external membrane is regulated by a calcium pump (calcium-magnesium-ATPase), calcium channels and binding to the membrane. In cell membranes and in lymphocytes of essential hypertensives, our group showed increased calcium and decreased magnesium and an increased calcium/magnesium ratio in hypertensive cells. In this context, in aortic smooth muscle cells from 13 spontaneously hypertensive rats (SHR) of the Münster strain (systolic blood pressure 188.4+/-9.8 mmHg) and 13 normotensive rats (NT, systolic blood pressure 118.5+/-7.2 mmHg) aged 9 months, the intracellular calcium and magnesium contents were measured under nearly in vivo conditions by electron-probe microanalysis. Measurements were performed in aortic cryosections 3 microm thick. The calcium content was 124.7+/-4.5* mmol/kg dry weight in SHR versus 110.3+/-4.1 mmol/kg dry weight in NT (Means+/-SD, p < 0.01), the magnesium content was 35.5+/-3.9* in SHR versus 50.1+/-4.9 mmol/kg dry weight in NT /p < 0.01). The calcium/magnesium ratio was significantly increased in SHR versus NT (3.56+/-0.39* versus 2.23+/-0.27, p < 0.01). In hypertensive one month old animals the increase in the calcium/magnesium ratio was not as pronounced as in 9 month old animals. The calcium/magnesium ratio was measured 3.3+/-0.42 in SHR (n = 8) as compared to 2.51+/-0.39 in normotensive animals (n = 8, p < 0.01). Aortic smooth muscle cells from SHR are characterized by markedly elevated intracellular calcium and decreased intracellular magnesium contents compared with normotensive cells. The increased calcium/magnesium ratio in hypertensive cells may be a pathogenetic factor for the development of arteriosclerosis and hypertension.     

Plasma ammonia and liver ornithine transcarbamoylase activity in zinc-deficient rats

The effects of zinc deficiency on the activity of hepatic ornithine carbamoyltransferase (OCT) and plasma ammonia were studied in rats. One group received (ad libitum) zinc-deficient diet containing 2 ppm zinc and the other group received a diet containing 110 ppm zinc (group pair-fed control) equal to the amount consumed by zinc-deficient rats during the previous 24 h. Rats were killed at weekly intervals. Blood urea nitrogen (BUN), plasma ammonia, and hepatic OCT activity were determined. By end of the 1st wk on zinc-deficient diet, the plasma ammonia levels became significantly higher than those of the controls and remained elevated thoughout the study period. BUN increased initially for 2 wk in the deficient rats, but by the end of 4 wk the levels were lower than in the controls. The hepatic OCT activity in deficient animals was significantly lowered as compared to the controls by the 3rd wk. It is concluded that an increase in plasma ammonia may occur as a result of deficiency of zinc.     

Behavioral characteristics of rats experiencing chronic zinc deficiency.

Adult rats chronically deprived of dietary zinc do not behave as “hippocampal” animals despite evidence that the deficiency alters the electrophysiological properties of normally zinc-rich hippocampal mossy fibers. The behavioral characteristics of these animals differed from controls and were substantially parallel to those reported for animals with excess glucocorticoids. Since zinc deficiency has been reported to produce a hyperadrenal condition this raises the possibility that the behavioral abnormalities that accompany zinc deficiency may be mediated largely by alterations in production and/or utilization of glucocorticoids.     

Vitamin D deficiency reduces the benefits of progesterone treatment after brain injury in aged rats

Administration of the neurosteroid progesterone (PROG) has been shown to be beneficial in a number of brain injury models and in two recent clinical trials. Given widespread vitamin D deficiency and increasing traumatic brain injuries (TBIs) in the elderly, we investigated the interaction of vitamin D deficiency and PROG with cortical contusion injury in aged rats. Vitamin D deficient (VitD-deficient) animals showed elevated inflammatory proteins (TNFα, IL-1β, IL-6, NFκB p65) in the brain even without injury. VitD-deficient rats with TBI, whether given PROG or vehicle, showed increased inflammation and greater open-field behavioral deficits compared to VitD-normal animals. Although PROG was beneficial in injured VitD-normal animals, in VitD-deficient subjects neurosteroid treatment conferred no improvement over vehicle. A supplemental dose of 1,25-dihydroxyvitamin D₃ (VDH) given with the first PROG treatment dramatically improved results in VitD-deficient rats, but treatment with VDH alone did not. Our results suggest that VitD-deficiency can increase baseline brain inflammation, exacerbate the effects of TBI, and attenuate the benefits of PROG treatment; these effects may be reversed if the deficiency is corrected.     

The effect of zinc deficiency on the ultrastructure of rat adrenal cortex

The adrenal cortex of male Sprague-Dawley weanling rats maintained on a zinc-deficient diet was studied by electron microscopy. There was a gradual increase in lipid droplets in the zona fasciculata which was quite marked at 7 weeks, but increased lipid was not observed in the zona glomerulosa or reticularis of zinc-deficient rats. The adrenal cortex of the control animals did not show altered lipid content. A possible explanation for the effect of zinc deficiency on the adrenal cortex is offered.     

Enhanced contractile response to noradrenaline and calcium influx in thoracic aorta isolated from dietary magnesium deficient rats

Dependency on extracellular calcium influx in contractile response to noradrenaline has been investigated in the thoracic aorta isolated from dietary magnesium (Mg) deficient rats. Adult male Wistar rats were fed with a Mg deficient diet (0.001% Mg) for 30 days, together with control groups (0.07% Mg). The contractile response to noradrenaline in the thoracic aortas from Mg deficient rats was significantly greater than that in the controls. However, there were no significant differences between control and Mg deficient rats in the contractile response to high potassium. The rate of 45Ca uptake induced by noradrenaline was greater in Mg deficient rat aortas than in the controls, but that induced by high potassium showed no significant differences between control and Mg deficient rats. Calcium entry blockers (verapamil and nifedipine) caused a concentration-dependent depression of the noradrenaline-induced contraction. The aortas from Mg deficient rats were more sensitive to these drugs than were those from the controls. These results suggest that the noradrenaline-induced contraction depends much more on extracellular calcium influx in Mg deficient rats than in the controls.     

Regulation of renal adenylate cyclase by parathyroid hormone

This study reports the effects of the removal of endogenous PTH by thyroparathyroidectomy (TPTX) on the recovery of the reduced renal cAMP response to parathyroid hormone (PTH) in rats with chronically elevated PTH secondary to diets deficient in either vitamin D or calcium. After TPTX and infusion with a calcium-glucose solution of the vitamin D-deficient rat, calcium and PTH fell from 5.8 mg/dl and 2,509 pg/ml, respectively, to 4.8 mg/dl and 160 pg/ml at 48 h. There was a partial restoration of response to PTH, assessed by assay of renal cortical adenylate cyclase activity from 64% of control activity prior to TPTX to 84% of control activity at 48 h. When rats fed the diet deficient in calcium were TPTX, serum PTH fell rapidly from 2,811 to 200 pg/ml at 5 h with no further change at 21 h, whereas calcium did not change (5.3 mg/dl). PTH-dependent adenylate cyclase activity increased from 59% of control activity prior to TPTX to 87% at 5 h and 100% of control activity at 21 h after TPTX. Each diet produced similar increases in the serum level of immunoreactive PTH, and the rate of disappearance of the circulating hormone after TPTX was also similar for both groups of rats. The data indicate a slow, partial recovery of the enzyme response to PTH after TPTX of the vitamin D-deficient rat over the time period studied, whereas the recovery was rapid and complete in rats fed the diet deficient in calcium.     

VITAMIN A DEFICIENCY INCREASES INFLAMMATORY RESPONSES

The authors studied the influence of vitamin A deficiency on immediate and delayed type hypersensitivity as well as granulocyte-mediated inflammatory reactions in vitamin A depleted and control rats. The number of circulating leucocytes was 43% higher in the vitamin A deficient than in the control animals. The leucocytosis was a result of a general increase of white blood cells and was not due to an increase in one particular type. The ratio between CD4⁺ and CD8⁺ T cells was unchanged. The vitamin A deficient rats had a four times higher T-cell proliferative response and a two times higher interferon-γ production in vitro than the control animals. In accordance, the DTH reaction was consistently higher in the vitamin A deficient rats. The granulocyte dependent inflammation, induced by olive oil injection, was also strongly enhanced in the vitamin A deficient rats compared with the controls. In addition, the spontaneous release of nitric oxide from the peritoneal phagocytes was five times higher in the vitamin A deficient animals. The number of peritoneal mast cells was about one and a half times higher in the vitamin A deficient than in the control animals. The density of IgE-receptors on the mast cells, the IgE receptor occupancy and the histamine release from the mast cells did not differ between the groups, however. The vitamin A deficient immunized rats displayed a consistently stronger immediate skin reaction after intracutaneous antigen injection than the immunized control rats, despite lower IgE antibody levels. The skin reaction after intracutaneous injection of histamine was also significantly greater in the deficient animals. Despite the stronger reaction to antigen and histamine, the passive cutaneous anaphylaxis reaction was lower in the vitamin A deficient rats. In conclusion the study shows that vitamin A deficiency aggravates the clinical manifestations of inflammatory reactions. Thus, vitamin A deficiency might lead to a higher risk of acquiring irreversible tissue damage and disabling destruction.     

The alteration of magnesium, calcium and phosphorus metabolism by dietary magnesium deprivation in postmenopausal women is not affected by dietary boron deprivation.

A study with human volunteers was conducted to test the hypothesis that naturally occurring inadequate intakes of magnesium induce negative magnesium balance and undesirable changes in calcium metabolism variables, and that these changes are influenced by dietary boron. Diets composed of ordinary Western foods providing approximately 118 and 318 mg Mg/d and approximately 0.25 and 3.25 mg B/d were fed in a double-blind Latin square design to 13 healthy, post menopausal Caucasian women (aged 50-78 years) living in a metabolic unit. Magnesium balance, which was positive when dietary magnesium was 318 mg/d, became negative when dietary magnesium was 118 mg/d. Magnesium deprivation decreased urinary calcium excretion, and significantly increased calcium balance when balance data analyzed came from all collections during the 42-day periods. Urinary phosphorus excretion was increased, but fecal phosphorus excretion was decreased, thus phosphorus balance was not significantly affected by magnesium deprivation. Magnesium deprivation did not affect manganese or zinc balance. The balance data indicated that 700 mg of calcium, 1.0 mg of manganese, and 10 mg of zinc were adequate for post menopausal women. Magnesium deprivation increased serum 25-hydroxycholecalciferol and decreased serum total cholesterol concentrations. Boron deprivation increased but magnesium deprivation decreased urinary potassium excretion. Boron supplementation decreased serum 17beta-estradiol and progesterone when dietary magnesium was low. The dietary treatments did not affect serum calcitonin, parathyroid hormone, osteocalcin or alkaline phosphatase concentrations. One woman placed on consecutive magnesium-low dietary periods exhibited heart ventricular ectopy after consuming the magnesium-low diet for 72 days; the ectopy disappeared upon consuming the magnesium-adequate diet. The findings indicated that consuming an ordinary diet deficient in magnesium, resulting in negative magnesium balance, can affect calcium, potassium, and cholesterol metabolism. Dietary boron did not have an obvious effect on the response to magnesium deprivation.     

缺锌下调3周龄小鼠肾脏凋亡相关因子BCL-2表达

 目的 观察生长期小鼠锌缺乏条件下肾脏细胞凋亡相关因子Bcl-2表达的变化,进而探讨锌离子对肾细胞凋亡的影响。 方法 根据小鼠精神状况、摄食量、体质量增长及血清锌含量变化确定模型构建是否成功。用免疫荧光和蛋白印迹技术检测肾组织中Bcl-2的表达。结果 缺锌小鼠出现典型缺锌症状,体质量下降,摄食量减少,血清锌含量明显降低（P<0.05）。在肾脏Bcl-2定位表达于肾小管和集合管上皮细胞胞质内,锌缺乏小鼠肾脏Bcl-2的表达量明显低于对照组（P<0.05）。结论 生长期锌缺乏可使肾组织中Bcl-2 蛋白表达下调,有可能促进肾细胞凋亡的发生。     

The role of the endogenous opiates in zinc deficiency anorexia

Anorexia is a major symptom of zinc deficiency, but the mechanism(s) for this anorexia are poorly defined. Recent studies have suggested an integral role for endogenous opiate peptides in appetite regulation. Dynorphin, a leucine-enkephalin containing opiate peptide, is a potent inducer of spontaneous feeding. In this study we showed that zinc deficient animals were relatively resistant to dynorphin-induced feeding. Measurement of dynorphin levels using a highly sensitive radioimmunoassay showed that zinc deficient animals had lower levels of dynorphin in the hypothalamus than did ad lib fed animals, with weight restricted animals having intermediate values. [3H]-naloxone binding was significantly increased to isolated brain membranes from zinc deficient animals using 1 nM unlabeled naloxone when compared to ad lib fed controls with the weight restricted animals again having intermediate values. These data suggest that abnormalities in endogenous opiate regulation of appetite may well play a role in the anorexia of zinc deficiency. The effects of zinc deficiency on endogenous opiate action appear to include alterations in receptor affinity, a post-receptor defect and alterations in the synthesis and/or release of dynorphin.     

Significance of serum levels of vitamin D and some related minerals in breast cancer patients

Vitamin D and calcium are involved in a wide range of proliferation, apoptosis and cell signaling activities in the body. Suboptimal concentrations may lead to cancer development. The role of phosphate in cancer metabolism is particularly relevant in breast cancer while, magnesium deficiency favors DNA mutations leading to carcinogenesis. Objectives: To determine serum levels of vitamin D, calcium, phosphorus, magnesium, and parathormone in female breast cancer patients and to assess their association with some prognostic factors in breast cancer. Design and methods: This study is done on 98 newly diagnosed female breast cancer patients and 49 age matched apparently healthy female volunteers as controls. Serum samples from all patients and controls were subjected to 25-OH Vit D, calcium, phosphorus, magnesium, and parathormone measurements. Results: In the breast cancer group, the median serum levels of 25-OH Vit D were 15 ng/ml, while it was 21 ng/ml in the control group. Levels of 25-OH Vit D and other tested minerals were significantly lower while calcium:magnesium (Ca:Mg) ratio, and calcium:phosphorus (Ca:P) ratio were significantly higher in the breast cancer group. Significant negative correlation was detected between phosphorus and calcium, ionized calcium , calcium magnesium ratio, and calcium phosphorus ratio. Conclusion: It is not only the deficient levels of Vit D and other related minerals, but the combination of the abnormal levels of all the studied parameters that might contribute to the development of cancer. Further studies with larger number of patient are needed.